Rat liver epithelial (RLE) cells provide a very valuable in vitro model to study both spontaneous and chemically induced hepatocarcinogenesis. Therefore, the main objectives of this project are to characterize the early cellular biochemical events as a result of genetic alterations and subsequent changes in protein expression during spontaneous and chemically mediated hepatocarcinogenesis. RLE cells can be transformed in vitro by various agents including chemical carcinogens and specific oncogenes as well as spontaneously when the cells are maintained under certain selective growth conditions. When injected into nude mice the transformed cells produce a diverse variety of tumor types including those of a well-differentiated hepatocellular carcinoma-like tumor. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was utilized to construct comprehensive computerized topographical protein database maps of protein expression in each of the major subcellular fractions (whole cell, cytosol, nuclei, mitochondria, plasma membrane). Genetic analysis of aflatoxin Bl induced RLE transformants revealed a consistent point mutation consisting of a transition from G to A within codon 173 (CGC to GAG) in the tumor suppressor p53 gene. This mutation resulted in a change of the encoded amino acid from histidine to arginine. Mutations in the p53 gene are currently being analyzed in monkey genomic DNA extracted from paraffin embedded AFB induced tumors.